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. 2011 Aug;79(8):3438–3444. doi: 10.1128/IAI.01252-10

Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †

Markus O Henke 1,*, Gerrit John 1,2, Christina Rheineck 1, Shashi Chillappagari 1, Lutz Naehrlich 3, Bruce K Rubin 4
Editor: B A McCormick
PMCID: PMC3147599  PMID: 21646446

Abstract

Airway mucins are the major molecular constituents of mucus. Mucus forms the first barrier to invading organisms in the airways and is an important defense mechanism of the lung. We confirm that mucin concentrations are significantly decreased in airway secretions of subjects with cystic fibrosis (CF) who have chronic Pseudomonas aeruginosa infection. In sputum from CF subjects without a history of P. aeruginosa, we found no significant difference in the mucin concentration compared to mucus from normal controls. We demonstrate that mucins can be degraded by synthetic human neutrophil elastase (HNE) and P. aeruginosa elastase B (pseudolysin) and that degradation was inhibited by serine proteases inhibitors (diisopropyl fluorophosphates [DFP], phenylmethylsulfonyl fluoride [PMSF], and 1-chloro-3-tosylamido-7-amino-2-heptanone HCl [TLCK]). The mucin concentration in airway secretions from CF subjects is similar to that for normal subjects until there is infection by P. aeruginosa, and after that, the mucin concentration decreases dramatically. This is most likely due to degradation by serine proteases. The loss of this mucin barrier may contribute to chronic airway infection in the CF airway.

INTRODUCTION

Mucus is the first barrier defense of the airways protecting against invading organisms (12). Mucus is mainly composed of water and ions (95%), with proteins and lipids secreted by airway epithelial cells making up most of the remaining mass (8). In health, mucin glycoproteins are the major macromolecular component of the mucus (36). Mucins are responsible for the rheologic and barrier properties of mucus. The viscoelasticity of mucus depends on mucin hydration and cross-linking and on the mucin concentration (48). Invading organisms are bound to mucins by their alternating hydrophilic and hydrophobic regions, and bonds are developed between the pathogen and the flexible fibers of the mucins in the mucus gel (9) (for a review, see reference 11). The flexibility of mucin oligomers enables the mucus to stick to surfaces and form multiple low-affinity bonds. Just as polyvalent antibodies, such as IgM, can bind to a pathogen with high “avidity” by making multiple lower-affinity bonds, the mucins make numerous low-affinity bonds to the pathogens and trap them within the mucus gel to be transported out of the airways via the mucociliary escalator or by cough.

There are two major classes of mucins: secreted and membrane-tethered mucins (36). In sputum, MUC5AC and MUC5B are the major oligomeric gel-forming, secreted mucins, with trace amounts of MUC2 (24). MUC5AC appears to be produced primarily by goblet cells in the tracheobronchial surface epithelium, and MUC5B is secreted primarily by submucosal glands (47).

Sputum is a product of airway inflammation and usually contains cells, inflammatory mediators, bacteria, and polymerized DNA from inflammatory cell necrosis (38). Chronic airway diseases, such as asthma, chronic bronchitis (CB), and cystic fibrosis (CF), are thought to be associated with mucus hypersecretion. Sputum from subjects with CB contains less MUC5AC than normal mucus (43). We reported that the mucin concentration from adult CF subjects with chronic Pseudomonas aeruginosa infection was significantly decreased compared to that from healthy controls (20). In adult CF subjects with chronic P. aeruginosa infection, airway mucin increases at the start of a pulmonary exacerbation but only to the concentration found in healthy controls (19).

Although structurally the lungs of CF patients appear normal at birth (13), there is a neutrophil-dominant inflammatory response in the airways with increased production of cytokines, including interleukin 8 (IL-8) and IL-6 (30). This inflammatory response is associated with increased concentrations of proteases in the airway surface liquid (ASL), which overwhelms antiprotease capacity (7). Proteases have multiple functions (44). The neutrophil-related proteases have antimicrobial properties in vivo (5). During an acute inflammatory response, these enzymes are released into the extracellular environment, where they play important roles in proteolytic processes (2). They further can induce apoptosis of epithelial cells via activation of surface receptors, such as protease-activated receptor 1 (PAR-1) (42). Human neutrophil elastase (HNE) can also degrade components of the pulmonary extracellular matrix, including elastin, type I to IV collagens, proteoglycans, fibronectin, and laminin (16).

The potentially destructive action of proteases in bronchial secretions is primarily controlled by two antiproteases: α1-antitrypsin (α1-AT) and secretory leukoprotease inhibitor (SLPI), a cationic protein found in serous secretory glandular cells (32). In healthy airways, antiproteases are present in higher quantities than proteases and provide a protease screen (6).

Decreased mucin in the CF airway could be related to the CF transmembrane ion regulator protein (CFTR) defect, which results in an elevated cytosolic pH. A defect in the Golgi pH in CF cells could decrease the activity of pH-sensitive enzymes, which might alter intracellular glycoprotein (mucin) processing (4). A further possibility based on the CFTR defect could be an altered HCO3− secretion. In affected organs, mucins tend to remain aggregated, poorly solubilized, and less transportable (33). Decreased mucin concentrations could also be a result of chronic P. aeruginosa infection in CF airways. The mucins might be consumed by P. aeruginosa or degraded by enzymes released by bacteria and inflammatory cells, leading to a diminished protective shield and possibly improved growth conditions for bacteria. A third hypothesis could be that the associated inflammatory response causes a diminished functional airway epithelium that is less capable of producing and secreting mature mucins for maintaining airway protection.

We wanted to determine if the reduced CF airway mucin concentration was related to the CFTR defect or to chronic P. aeruginosa infection. Therefore, we investigated mucin concentrations in CF subjects without any documented P. aeruginosa colonization and compared this with findings for CF subjects with intermittent and chronic P. aeruginosa infection. We also evaluated the effect of proteases on airway mucin in vitro.

MATERIALS AND METHODS

Subjects.

Sputa were collected from patients who attended the CF clinic of the University of Giessen at least every 3 months. Sputum was collected by direct expectoration, without the use of saline induction during a 45-min pulmonary physical therapy training as part of the program of the CF clinic. Bacteriological analysis of the sputum was routinely performed by quantitative bacterial culture in the clinical laboratory of the Justus-Liebig University Giessen. The clinical characteristics and demographics of the CF subjects are given in Tables S1 and S2 in the supplemental material.

Sputum collection was approved by the Justus-Liebig University Giessen Institutional Review Board; patients signed informed consent. Endotracheal tube (ETT) mucus was collected and approved by the Wake Forest University Institutional Review Board (by BKR) and signed consent, and assent was obtained when appropriate.

The subjects were classified as follows: group 1, patients were considered to have “no colonization” if neither P. aeruginosa nor Burkholderia cepacia complex (BCC) was detected in sputa or pharyngeal swabs in 8 longitudinal and sequential sputum collections during clinic or hospital visits; subjects were excluded if we could not document 8 consecutive sputa with no Gram-negative pathogens; group 2, patients were considered to have an “intermittent infection” if 1 to 4 sputum samples were positive for either P. aeruginosa or BCC in 8 sequential sputum collections (over a minimum of 2 years); group 3, patients were considered to have “chronic infection” if P. aeruginosa was detected in 3 consecutive sputa or in more than 5 sputa of 8 sequential sputum collections over a minimum of 2 years; group 4, patients were considered to have an “exacerbation” if they had signs of increased dyspnea, fever, weight loss, increased cough, increased sputum production, hypoxemia, and a decrease in weight or exercise tolerance, along with a documented decrease in FEV1 of at least 5% from the previous clinic visit in the preceding 3 months; any subject who had an exacerbation within the previous 3 months was categorized into this group; group 5, “control” mucus was collected from the ETT of subjects who had no lung disease and required nonthoracic surgery under general anesthesia. At the time the subject was extubated, the ETT was removed from the airway and mucus coating the tube was removed by gently scraping the ETT (37, 39).

MUC5AC and MUC5B antibodies.

Polyclonal anti-MUC5AC and anti-MUC5B antibodies were generated as previously described (20). The antibodies were characterized and specificity was ascertained by preabsorption studies using increasing concentrations of the antigenic peptides (21). To verify the specificities of our antibodies, we performed a PAGE with cell lysates, secretions from normal human tracheobronchial epithelial (NHTBE) cells (passage 2) (Clonetics Corp., La Jolla, CA), and human mucus. The blots were analyzed with antisera for MUC5AC and MUC5B and the preimmune sera of the same rabbit. We found one well-defined band of high molecular weight with the antisera. To increase the specificities of the antibodies and reduce nonspecific binding, we affinity purified the antipeptide antibody from the whole serum using the immobilized amino acid sequences of interest (SulfoLink kit; Pierce).

Preparation of internal control.

To compare different blots, we established an internal mucin control as previously described (20). The mucin control was collected from a voluminous sputum sample from a single patient undergoing lung transplantation for non-CF bronchiectasis. The control was applied twice to every blot at the same volume. The test samples were compared to the control to allow comparison of the samples on different blots. Mucin quantities in CF sputum and normal control mucus were normalized to this internal control (set at 100%).

Gel electrophoresis: agarose gel.

Sputum and internal control samples were applied in Laemmli buffer (250 mM Tris [pH 6.8], 4% SDS, 20% glycerol, 0.001% bromophenol blue, 20 mM dithiothreitol [DTT]) and electrophoresed in 1% agarose gels (15 by 15 cm), prepared in running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). Electrophoresis was performed in a horizontal gel apparatus at 100 V at room temperature. To identify small proteins that remained in the gel, the gel was stopped when the dye front was 2/3 of the distance from the wells. After electrophoresis, proteins were transferred to nitrocellulose membranes by electrical transfer (300 mA) for 3 h at 4°C.

Probing the blots.

The membranes were blocked with 5% nonfat skimmed milk in phosphate-buffered saline (PBS) for 30 min at room temperature. They were incubated with primary antibodies (1:100 MUC5AC; 1:250 MUC5B) for 1 h in 1% nonfat skimmed milk in PBS, washed 3 times in PBS for 10 min, and incubated with the secondary horseradish peroxidase (HRP)-labeled goat-anti-rabbit antibody (1:1,000; Jackson-Immuno) in 1% nonfat skimmed milk in PBS for 1 h. Finally, they were washed in PBS for 10 min, 3 times. Membranes were developed using the Pico developer kit (Pierce). Exposures were taken on X-Omat Blue XB-1 film (Kodak) at equal exposure times. The film was scanned, and band densities were determined by densitometry using the NIH Image software program (http://rsbweb.nih.gov/nih-image/).

Quantification of DNA in sputum.

DNA was measured by microfluorimetry and compared with a calf thymus DNA standard (Sigma, St. Louis, MO) at 0.25 to 10.0 μg/ml (22). The supernatant was diluted 1:50 for CF and 1:10 for ETT specimens with SSC solution (0.0154 M NaCl–0.015 M Na3-citrate, pH 7.0). One microliter of 33258 Hoechst (1.5× 10−6 M; (Calbiochem, La Jolla, CA) was added to the sample, and fluorescence was measured by spectrophotofluorimetry with an excitation wavelength at 360 nm and emission at 450 nm (22).

Mucin degradation in vitro and by proteases with and without protease inhibitors.

In order to measure the effect of mucin degradation in vitro, we incubated freshly collected sputum and mucus (without protease inhibitors) from CF subjects and normal controls at 37°C at 0, 1, 2, 3, 6, and 24 h. The experiment was terminated at the desired time points by adding protease inhibitors and freezing the sample immediately at −70°C until analyzed. Samples were evaluated by Western blotting.

To analyze mucin degradation by proteases, we incubated mucus from normal controls (ETT) with the synthetic proteases HNE and Pseudomonas elastase B (pseudolysin) for 3 h at 37°C. HNE (Merck Chemical, Nottingham, United Kingdom) and Pseudomonas elastase B (Merck Chemical, Nottingham, United Kingdom) were used as suggested by the manufacturer.

To assess whether mucin degradation in the CF sputum could be inhibited by a protease inhibitor, we incubated the sputum at 37°C over 6 h in the absence or presence of protease inhibitors. The serine protease inhibitors diisopropyl fluorophosphates (DFP), phenylmethylsulfonyl fluoride (PMSF), and 1-chloro-3-tosylamido-7-amino-2-heptanone HCl (TLCK), metalloprotease (EDTA and GM6001), and cysteine proteases (leupeptin and E64) were used. Chemicals were purchased from Sigma (St. Louis, MO) (DFP [final concentration, 2 mM], PMSF [final concentration, 2 mM], TLCK [final concentration, 10 mM], EDTA [final concentration, 100 mM], and E64 [final concentration, 500 ng/ml]) or Merck Chemical (Nottingham, United Kingdom) (GM6001 [final concentration, 40 μM] and leupeptin [final concentration, 40 μM]).

Data analysis.

Results are presented as mean values ± standard errors. The mucin concentration of the sputum samples was normally distributed within all groups (Kurtosis and Skewness < 2). After post hoc correction for multiple comparisons, a probability P value of <0.05 was considered significant. To compare sputum samples of the different groups, we used the Mann-Whitney U test. All analyses were performed by means of the GraphPad Prism 5 software program (San Diego, CA). Descriptive statistics were used to summarize subjects' demographics.

RESULTS

Mucin concentration in CF sputum and normal mucus.

Collected CF sputa were obtained from 52 subjects, grouped as follows: “no colonization” (n = 9), “intermittent infection” (n = 21), “chronic infection” (n = 6), and “exacerbation” (n = 16). We also collected ETT mucus from 11 control subjects. All samples were loaded on the gel as volume equivalents from the sputum. Because the avidities of the antibodies for MUC5AC and MUC5B were different, these separate results cannot be directly compared.

Data are from individual sputum samples analyzed in triplicate. All Western samples were normalized to an internal control as described above. To show a representative blot for the different CF groups 1 to 4, we included Fig. 1A. Due to the heterogeneity of the mucins, multiple bands can appear, as previously described (20, 24, 40).

Fig. 1.

Fig. 1.

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(A) CF sputum electrophoresis by Western blotting on a 1% agarose gel, blotting to nitrocellulose membranes, and probing with MUC5AC (left) or MUC5B (right) antibody. For patient details, see Table S1 in the supplemental material. The internal standard was used to compare different blots. Group 1, “no colonization”; group 2, “intermittent infection”; group 3, “chronic infection”; group 4, “exacerbation”; and normal ETT control. (B) Mucin concentration. Sputum from 9 subjects with CF who had no positive culture for P. aeruginosa or BCC over the last 8 sequential clinic sputum collections (CF, no colonization), 21 CF subjects who had 1 to 4 positive cultures for P. aeruginosa or BCC over at last 8 sequential clinic sputum collections (CF, intermittent infection), 6 CF subjects who had persistent P. aeruginosa or BCC cultures in sequential clinic sputum collections over at last 8 sputum analyses over at least 2 years (CF, chronic infection), and 16 subjects during an acute pulmonary exacerbation with positive P. aeruginosa or BCC culture (CF, exacerbation), compared with mucus from 11 normal ETT control subjects (control). The results are shown as mean density of individual samples related to the internal control (= 100% relative concentration). *, significant in comparison to “CF, no colonization” (P < 0.05); #, = significant in comparison to “control” (Mann-Whitney U test, P < 0.05).

In the “no colonization” group, the MUC5AC and MUC5B concentrations were 84% and 79% of our internal control mucus, compared with 94% and 71% concentrations for normal ETT control mucus (P value not significant [NS]) (Fig. 1B). There was also no significant difference when comparing MUC5AC and MUC5B concentrations in the “no colonization” group to those in the “intermittent” group (73% and 67%) (Fig. 1B). There was significantly more MUC5AC and MUC5B in the “no colonization” group than in the “chronic infection” group (40% and 46%) (Fig. 1B). There was also 21% less MUC5AC and 4% less MUC5B in the “intermittent infection” group than in the ETT control group, but this difference was not statistically significant (P = 0.09 for MUC5AC; P = 0.25 for MUC5B). In the “chronic infection” group, there was also significantly less MUC5AC (58%) and MUC5B (36%) than in the “control” group (Fig. 1B).

MUC5AC and MUC5B concentrations during a CF exacerbation were not significantly different (P = 0.40) from those in the ETT controls (Fig. 1B). These results are consistent with previously published results (19, 20).

DNA concentration in sputum from CF and in ETT control mucus.

The sputum DNA concentration was significantly lower for CF subjects without a history of P. aeruginosa colonization (1.16 mg/ml), than for chronically infected subjects (4.16 mg/ml) without evidence of a pulmonary exacerbation (P < 0.05) (Fig. 2). The DNA concentrations were not significantly different between the no-colonization and the intermittent-infection groups (P = 0.42) and the exacerbation groups (P = 0.10).

Fig. 2.

Fig. 2.

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Total DNA concentrations for subjects grouped as described the in text and the legend to Fig. 1B. CF, no colonization, n = 9; CF, intermittent infection, n = 21; CF, chronic infection, n = 6; CF, exacerbation, n = 16; normal controls, n = 11. *, significant in comparison to normal mucus (control); #, significant in comparison to “no colonization” (P < 0.05).

DNA levels were significantly lower in both normal mucus (0.85 mg/ml) and sputum from subjects with intermittent infection (2.37 mg/ml) than in sputum from chronically infected subjects (4.16 mg/ml).

Degradation of mucins over time in vitro.

We incubated sputa at 37°C at different time points (0 h, 1 h, 2 h, 6 h, and 24 h) and analyzed the mucin concentration by Western blotting in order to evaluate possible mucin degradation by endogenous proteases (Fig. 3). For mucus from normal ETT controls and sputum from CF subjects without a history of P. aeruginosa colonization, after 24 h of incubation approximately 50% of MUC5AC and MUC5B were still detectable. For these sputa, degradation was apparent by 3 h of incubation.

Fig. 3.

Fig. 3.

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Analysis of MUC5AC and MUC5B by Western blotting in sputum incubated at 37°C for 1, 2, 3, 6, and 24 h. Sputum was obtained from normal controls (ETT control), from CF subjects without any history of P. aeruginosa colonization (CF, no colonization), and from CF subjects with chronic P. aeruginosa infection (CF, chronic infection). After 24 h of incubation, mucin was detectable only in control mucus and in sputum from CF subjects without a history of P. aeruginosa colonization.

However, after 24 h of incubation, sputa from subjects with chronic P. aeruginosa infection had almost no detectable MUC5AC and MUC5B mucin, and MUC5B appeared to be fully degraded after just 3 h.

Mucin degradation by proteases.

We incubated mucus from healthy ETT controls for 3 h at 37°C with and without synthetic HNE and P. aeruginosa elastase B (pseudolysin) (Fig. 4). Both MUC5AC and MUC5B mucins were degraded by HNE (a serine protease) and by P. aeruginosa elastase B (a metalloprotease).

Fig. 4.

Fig. 4.

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Normal ETT control mucus was incubated for 3 h at 37°C with or without HNE and Pseudomonas elastase B (pseudolysin). The mucin concentration of the native control was set to 100%.

We then incubated CF sputum with inhibitors for serine protease (DFP, PMSF, and TLCK), inhibitors for metalloprotease (EDTA and GM6001), and inhibitors for cysteine proteases (leupeptin and E64). Incubation of sputum from a chronically infected subject at 37°C for 6 h reduced MUC5AC by 85% and MUC5B by 88% of the initial concentration. The serine proteases inhibitors (DFP, PMSF, and TLCK) inhibited mucin degradation, but metalloprotease inhibitors (EDTA and GM6001) and cysteine protease inhibitors (leupeptin and E64) did not (Fig. 5).

Fig. 5.

Fig. 5.

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Analysis of sputum MUC5AC and MUC5B by Western blotting after incubation at 37°C over 6 h in the presence or absence of proteases inhibitors. Sputum was obtained from a CF subject chronically infected by P. aeruginosa. The mucin concentration of the native control without incubation was set to 100%, and results are presented relative to this. We used the serine protease inhibitors DFP, PMSF, and TLCK, the metalloprotease inhibitors EDTA and GM6001, and the cysteine protease inhibitors leupeptin and E64.

DISCUSSION

Patients with CF have plugging of their airways by secretions, leading to increased susceptibility to infection and decreased pulmonary function. Although there is plugging and chronic sputum expectoration in persons with CF, mucin appears to be a minor component of these secretions and DNA is the dominant polymer. We previously showed that the amount of DNA in sputum from adult CF subjects was greatly increased over that in chronic bronchitis sputum (20), most likely as a result of leukocyte necrosis (31). In our current study, we found that mucin and DNA concentrations in sputum from CF subjects without a history of P. aeruginosa colonization are similar to those in normal control mucus and that while intermittent and chronic P. aeruginosa infection decreases mucins, DNA concentrations increase.

At the start of a pulmonary exacerbation, even those patients with chronic P. aeruginosa infection have a significant increase in mucin protein concentrations: MUC5AC increased by 118% and MUC5B increased by 57% relative to a period of stable disease. This suggests that the capacity to secrete mucin is intact in the CF airway, and we hypothesize that the acutely increased mucin concentration with an infectious exacerbation is a protective effect (19). This hypothesis is further supported by the finding that until there is chronic P. aeruginosa or BCC infection, there is no significant difference in the MUC5AC or MUC5B concentration in comparison with mucus from healthy subjects.

We showed that mucin can be degraded by the metalloprotease elastase B (also called LasB or pseudolysin) derived from P. aeruginosa and human neutrophil elastase (HNE), a serine protease derived from human leukocytes (23). Mucins from subjects with chronic P. aeruginosa infection degrade more quickly than mucins from CF subjects without P. aeruginosa colonization and from normal controls, suggesting a relative excess of endogenous proteases or a decrease in antiproteases in these airways. Adding serine protease inhibitors inhibits this degradation.

Many proteases are released by neutrophils, including HNE, protease 3, and cathepsin G. Serine proteases, HNE in particular, are present in high concentrations in CF airway surface liquid (ASL) (17). Neutrophils also release matrix metalloproteases (MMPs), such as MMP-8 and MMP-9, in CF ASL and bronchoalveolar lavage (BAL) fluid (34). Macrophages can release the cysteine protease cathepsin S and the metalloproteases MMP-1, MMP-9, and MMP-12 into the ASL. The cysteine proteases cathepsins B, L, and S (10) are also present in CF BAL (49).

Bacterial proteases are also present at high concentrations in the CF airway. The metalloproteases Pseudomonas elastase B, alkaline protease (AprA), and protease LasA (staphylolysin) are released by P. aeruginosa (41). P. aeruginosa also produces serine protease IV, which can degrade surfactant (1). These results suggest that P. aeruginosa and BCC are likely to be an important source of proteases. CF subjects without chronic P. aeruginosa infection but who had other protease-secreting bacteria cultured from their sputum had mucin concentrations that were comparable to those of normal ETT controls.

Vibrio cholerae produces a hemagglutinin/protease (Hap) which can hydrolyze mucin, fibronectin, and lactoferrin (14). By DNA sequence homology, Hap appears to be closely related to the Pseudomonas elastase B, a metalloprotease (28). Furthermore, it was shown that only P. aeruginosa elastase B-producing P. aeruginosa strains can degrade mucins (3). It has also been described that purified P. aeruginosa elastase B degrades ovomucin (18). In vitro, P. aeruginosa elastase B activity had a higher elastase-degrading activity than HNE (35).

HNE is a potent mucus secretagogue, inducing goblet cell degranulation and secretion of mucins from airway submucosal glands (for a review, see reference 15). HNE increases MUC5AC by transcriptional (26) and posttranscriptional (46) mechanisms. The neutrophil serine proteases cathepsin G and protease 3 can also stimulate mucus secretion, as do metalloproteases such as ADAM-10, meprin α, and MMP-9 (27, 29) and P. aeruginosa proteases, including Pseudomonas elastase B, alkaline protease, and protease IV (25). Cytokines including tumor necrosis factor alpha (TNF-α), IL-1β, IL-13, IL-6, and IL-17 upregulate MUC5AC expression (45). These mechanisms would be expected to increase CF airway mucin, presumably to protect the epithelium from damage. We show that CF subjects without P. aeruginosa colonization have mucin concentrations similar to those of normal controls, subjects with intermittent P. aeruginosa infection have decreased mucin concentrations, and those with chronic infection have profoundly decreased mucin. Our data suggest that this is due to a relative increase in serine proteases. Antiproteases may protect the airway in part by preventing mucin degradation. We hypothesize that by restoring mucin concentrations, bacteria will be trapped and cleared by mucociliary action and that the intact mucin barrier may inhibit the formation of bacterial biofilms.

ACKNOWLEDGMENTS

We thank Achim Boldt at the Justus-Liebig-University of Giessen for collecting the CF sputum samples during pulmonary physiotherapy. We thank Melissa Yopp at Wake Forest University and Virginia Commonwealth University for ETT specimens.

This study was supported by the Christiane Herzog Stiftung and by the Universities of Giessen and Marburg Lung Center (UGMLC) within the LOEWE program of the state of Hessen, Germany.

Footnotes

†

Supplemental material for this article may be found at http://iai.asm.org/.

▿

Published ahead of print on 6 June 2011.

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